JP2003530823A - Method for increasing the yield of recombinant protein in a microbial fermentation process - Google Patents

Abstract

  (57) [Summary] The use of prior art methods for the production of recombinant proteins in fed-batch fermentations often results in overgrowth of the culture with plasmid-free cells after induction of recombinant product synthesis, leading to a reduction in specific product yield . Thus, the yield of recombinant protein is increased by constantly reducing or increasing the concentration of the carbon / energy source in the culture over a short period of time. Vibration is created by varying the feed rate of the feed solution containing the carbon / energy source. This method is suitable for all microorganisms that are cultured using carbon-limited fed-batch.

Description

Detailed Description of the Invention

Production of recombinant proteins on an industrial scale in bacteria is carried out in fermenters. By increasing the amount of cells per volume, an increased yield is realized compared to laboratory scale experiments performed on a flask shaker. Batch fed technology can achieve high cell densities. It is based on the growth-limiting addition of nutrients that generally limit carbon / energy sources (eg, Riesenberg, D. and Guthk.
e, R.E. , 1999, App. Microbiol. Biotechnol. 5
1, 422-430). For the E. coli process, this is usually glucose or glycerol. Alternatively, however, depending on the microorganism and process used, other substrates such as molasses, starch, peptone, lactose, methanol and acetate are also used. The highly concentrated feed solution can be added continuously and various functions can be used to define the addition of substrate over a period of time or to linearly increase and decrease. Often combines various functions in a single step. Alternatively, the nutrient solution can be added in pulses (rhythmically) or at timed intervals, such that consumption of nutrients or a decrease in nutrient content below a given concentration serves as a signal for the next pulse (eg, Terasakawa et al., 1990, EP0 397 097 A1). Substrate solution addition can also be adjusted using other parameters. Dissolved oxygen (DO-stat), pH (pH-stat), or carbon dioxide and oxygen concentrations in the exhaust gas measured online (eg Kerns et al., Acta Biotech).
nol. 8, 285-289) can be used as control data to guide the periodic supply of nutrient solution. In doing so, the concentration of substrate varies between limiting and non-limiting concentrations. Chen et al. (1997, Biotechnol. Bioen.
g. 56, 23-31) measured the increase in plasmid stability when highly concentrated medium was periodically added to batch fed cultures. In these procedures, one cycle may last for minutes or hours, which has a detrimental effect on product formation.

In addition to the origin of replication, standard vectors for gene expression usually ensure that the DNA sequence encoding the desired protein (product gene) as well as the stable storage of the plasmid during culture growth. A plasmid containing a useful selectable marker. Expression of the product gene is usually controlled through regulatory sequences, especially regulatable promoters. The expression of the product gene is activated by, for example, changes in chemical inducers (substrate, substrate analog), culture temperature or other culture conditions (pH value, salt concentration, degree of substrate concentration). In particular, t by changing the restriction substrate or by lactose
Induction can also occur by induction of the ac promoter and switching from a glucose feed to a lactose feed (Neubauer et al., 1992, Appl.
Microbiol. Biotechnol. 36, 739-744). Genes that provide host cells with resistance to antibiotics serve as selectable markers for stable storage of plasmids in host cells. Therefore, in order to kill the plasmid-free cells that do not carry the resistance gene or inhibit their growth, the corresponding antibiotics are usually added to the culture for producing the recombinant protein. Commonly used resistance gene / antibiotic pairs are β-lactamase / ampicillin, chloramphenicol-acetyltransferase / chloramphenicol, tetracycline resistance (tet) -operon / tetracycline, and kanamycin resistance gene / kanamycin. .

Some of these resistance systems have the weakness that antibiotics are inactivated by resistance genes, as in the case of ampicillin and chloramphenicol (eg K
emp G.M. W. And Britz M .; L. , 1987 Biotechnol.
Techniques 1, 157-162). The effect of this inactivation is
That is, there is nothing that prevents the growth of plasmid-free cells in the culture.
In addition, resistance-mediating proteins may be released into the culture medium during pre-culture to promote antibiotic degradation. In these cases, the proportion of plasmid-free cells in the overall culture can be increased. Furthermore, in most industrial processes, trace amounts of residual antibiotic or its inactivated form must be removed, either for cost reasons or because of the extra costs associated with subsequent cleaning. No antibiotics are used. At the same time, some proportion of plasmid-free cells appear in such a process.

Although plasmid-free cells often have the advantage of low proliferation during the growth phase,
In many cases, after the product formation is initiated, there is a decrease in the growth rate of the plasmid-containing producer cells, which results in overgrowth of the culture by plasmid-free cells. The accumulation of plasmid-free cells has the drawback of reducing the relative proportion of products in the total cell number, making these post-fermentation steps more difficult, depending on the method of degradation and cleaning chosen.

When constructing the vector, for example through selection of resistance genes, use of alternative antibiotic independent stabilization systems (Molin and Gerdes, WO 84/0.
1172), or modified antibiotics that degrade more slowly, it is possible to limit these adverse effects, but again problematic resistance is used. Moreover, none of the alternative systems are infinitely stable; they can only remain stable for a period of time.

The invention as claimed in claim 1 provides for the overgrowth of plasmid-free cells after induction of recombinant product synthesis in batch fed-fermentation, especially in industrial applications, without negatively affecting product formation. Based on the challenge of restraint.

The features recited in claim 1 solve this problem by increasing / decreasing the concentration of the carbon / energy source in a periodic vibration pattern. This is achieved by varying the rate of addition of the feed solution containing the carbon / energy source, for example by corresponding programming of the pump to meter the feed solution. This leads to a successive phase in which the cells can use only a limited amount of substrate or none at all.

Contrary to previous belief that oscillations have a detrimental effect on product formation in the recombination process, the maximum cycle duration is 4 minutes and the individual phases of the cycle are targeted to last up to 2 minutes. Surprisingly, the vibrations have a positive effect on the product yield. A cycle period of about 1 minute (30 seconds feed, 30 seconds rest) is especially preferred.

The advantage of this method, which is basically applicable to all recombinant growth restriction steps in which the production of recombinant products is induced under carbon limitation, is that no additional substrate has to be added to the fermentation medium. Irrelevant to the expression system in which it is expressed and that it does not have a deleterious effect on the production of the product. This procedure is particularly suitable for batch fed processes in which sugars such as glucose, lactose, arabinose, or galactose, or other organic carbon sources such as methanol, glycerol, molasses, or starch as limiting nutrients. Add to medium. Such a procedure is independent of the culture medium and can be used for culturing in mineral salts medium as well as natural medium.

This method is not limited to Escherichia coli as a host organism, but may be Bacillus subtilis or brewer's yeast (
Saccharomyces cerevisiae), or Pichia p
It can be used with all microorganisms cultivated using a carbon restricted batch feed, such as astoris. This method is also independent of the induction system. But ta
It is particularly beneficial when using the c promoter.

Such a procedure is particularly beneficial when the expression of the gene product is strongly induced and the proliferation of producer cells is detrimentally affected compared to uninduced culture. In addition, this procedure
It is useful in processes with a particularly long production phase, for example in the periplasmic expression of recombinant proteins or when the product production phase is associated with temperature changes.

Operation method Strain and plasmid Escherichia coli K-12 RB791 (F ⁻ , IN (r
mD-rmE) 1, λ ⁻ , lacI ^q L _g ; coli Stock Cen
ter, New Haven, USA) was used as the host. This strain, S
The plasmid pKK177glucC (where the α-glucosidase gene from A. cerevisiae cerevisiae is under the control of the tac promoter.
Transformation with Kopetzki et al., 1989a). The plasmid contains the β-lactamase gene as its selectable marker. Furthermore, the plasmid pKK177
In addition to glucC, the dnaY gene (Minor-tRNA argU, AG
A / AGG) containing plasmid pUBS520 (Brinkmann et al., 198).
The second system transformed from 9) was used.

Culture medium and fermentation conditions Glucose-ammonium-inorganic salt medium (Teich et al., 1998, J. Bi
otechnol. 64, 197-210) was used for all cultures. The initial concentration of glucose was 5 g / l. The feed solution is 200 g glucose / k
g and all components of the medium at corresponding concentrations (exception: (NH ₄ ) ₂ SO ₄ 2.0
g / l) and 10 ml / l of trace element solution (Holme et al., 1970).
However, MgSO ₄ was not included. 10 ml of 1M MgSO ₄ solution during culturing
, OD ₅₀₀ = 9. Ampicillin (100 mg / l) and kanamycin (
10 mg / l) was added to both preculture and fermentation medium. Polypropylene glycol 2000 (50%) was used as the defoamer.

Shake cultures in fermented mineral salts medium grown at 37 ° C. were used as fermentation inoculum. All fermentations were run with 6 liters of Biostat ED Bioreactors.
At an initial volume of 4 L and a temperature of 35 ° C. The culture was started as a batch culture. At this stage, the aeration rate and agitation were adjusted stepwise to maintain a DOT of at least 20%. At the end of the batch stage, the DOT control was deactivated and the aeration rate and agitation speed was set to 2 vvm or 800 rpm. The pH value was adjusted to 7.0 using 25% ammonia solution. Approximately 2 g DCW / l at the end of the batch stage
At a cell density of (OD ₅₀₀ = 9), a feed pump was 53.2 g / hr (2.6 g).
It was started at a constant rate of glucose / l / h). The total amount of glucose added was the same in all cultures, regardless of feed method. Three different feeding strategies were investigated: (A) continuous feed (control culture), (B) intermittent feed with a 1 minute cycle (30 second feed, 30 second rest), (C) 4 minute cycle ( 2 minutes feed, 2 minutes rest) intermittent feed. 1 mM IPTG was added 3 hours after the feed was initiated to induce expression of the α-glucosidase gene and product formation was followed for approximately 20 hours after induction.

Analytical Method Cell proliferation was followed by measuring the optical density at 500 nm (OD ₅₀₀ ). The cell number was further measured under a microscope in a counting plate (depth 0.02 mm) to determine the dry cell weight (DCW) (Teich et al., 1998, J. Biot.
echnol. 64, 197-210). Colony forming units (c) by plating diluted samples on nutrient agar plates and incubating for at least 3 days.
fu) The number was measured. The stability of the plasmids was then investigated by pressing these plates onto selective agar by the replica plate method. _DCW, the relationship _{OD 500} and cell number following manner characterized: DCW of 1 g / l corresponding to _{OD 5 00} and 1.8 × ¹⁰ 9 / ml Number of cells 4.5 ± 0.1 To do. Glucose concentration was measured using a commercially available enzyme kit.

The α-glucosidase concentration was measured after separating whole cell samples on SDS gels (5% collecting gel, 7% separating gel). Product strips were scanned to perform expression and quantified relative to product standards placed in the gel at various concentrations.

Results E. coli RB791 pKK177glucC and E. coli. coli RB
791 pKK177glucC pUBS520 was cultivated in a stirred reactor using a glucose limited batch feed. After the first batch step, a constant feed was started, and 3 hours after the feed was started, 1 mM IPTG was added to induce the expression of the α-glucosidase gene. After induction, the α-glucosidase concentration rises, which causes the specific concentration of enzyme per cell to reach its maximum about 5 hours after induction and begins to decline in longer-term cultures (see FIG. 1c). The decrease in the α-glucosidase specific concentration is due to overgrowth of the culture by plasmid-free cells. Plasmid-free cells are highly advantageous for post-induction growth because the production of α-glucosidase has a detrimental effect on growth and also causes an inhibition of glucose uptake by the producing cells. The product gene-free cells present in culture are unaffected by the inducer IPTG and continue to grow endlessly due to the high availability of glucose.

When the glucose solution was not continuously added, but in pulses of short cycles with an interval of about 1 minute (see “Test materials and methods”), the α-as was the case with the constant feed after induction.
Glucosidase accumulates. However, overgrowth by plasmid-free cells is
It can be prevented depending on the duration of the pulse (see Figure 1d). This positive effect on the suppression of plasmid-free cells is not limited to the strong expression system shown in FIG. It was also apparent in the weak expression of α-glucosidase in the E. coli RB791 pKK177glucC system (FIG. 2, Table 1). Moreover, the pulse feed has a slightly positive effect on the synthesis rate in both cases after induction,
In the first case it also has a slight positive effect on the stability of the product,
% Or more was present in the form of inclusion bodies. The definition of cycle time is an important factor. In both experiments presented, extending the cycle time to 4 minutes leads to a reduction in the amount of product and thus a reduction in yield (see Figures 1, 2 and Table 1). Again, overgrowth of plasmid-free cells was reduced, but longer cycle times lead to reduced product synthesis or increased degradation.

[0019]

[Table 1]

[0020]   The drawings show:

FIG. 1 E. coli induced by 1 mM IPTG. coli RB791 pKK177
Batch fed fermentation with glucC pUBS520. Cycle addition of the same solution as continuous addition of glucose substrate solution (ac; white symbol: no induction; black symbol: with induction) (black triangle: 1 minute cycle; white triangle 4 minute cycle) )comparison. (A, d) Cell mass (DCW), (b, e) Glucose concentration, (c, f) Product production (mg α-glucosidase / g cell dry weight). The data presented show the characteristic fermentation of two experiments performed for continuous addition and one experiment each for cycle addition. The start time of the addition of the substrate solution (...) and the induction with IPTG were carried out 3 hours after the start of the feed (---).

FIG. 2 E. coli induced by 1 mM IPTG. coli RB791 pKK177
Batch fed fermentation for glucC. Continuous addition of glucose substrate solution (a-
c; white symbol: no induction; black symbol: induction, same cycle addition (d)
-F) (black triangle: 1 minute cycle; white triangle 4 minute cycle) comparison. (A, d)
Cell mass (DCW), (b, e) glucose concentration, (c, f) product production (mgα
-Glucosidase / g cell dry weight). See FIG. 1 for further explanation.

FIG. 3 shows a schematic diagram of a pump ball in fermentation in a 1-minute cycle. Dissolved oxygen (DO
T,%, -v-) reaction and pump switching (0 = off, 1 = on,-)
Together, it shows a small part of the fermentation.

[Brief description of drawings]

FIG. 1 E. coli induced by 1 mM IPTG. coli RB791 pKK177
1 shows the course of batch fed fermentation for glucC pUBS520.

FIG. 2. E. coli induced by 1 mM IPTG. coli RB791 pKK177
1 shows the course of batch fed fermentation with glucC.

[Figure 3]   FIG. 2 shows a schematic diagram of a pump ball in fermentation in a 1-minute cycle.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, C N, CR, CU, CZ, DE, DK, DM, EE, ES , FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, K R, KZ, LC, LK, LR, LS, LT, LU, LV , MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, S I, SK, SL, TJ, TM, TR, TT, TZ, UA , UG, US, UZ, VN, YU, ZA, ZW F-term (reference) 4B064 AG01 CA02 CA19 CC04 CC24                       CD04 CD06 CD07 CD09 CD19                       CD22                 4B065 AA26X AB01 BA02 BB06                       BB08 BB15 BB16 BB18 BB26                       BC18 BC50 CA24 CA31 CA60

Claims (9)

[Claims] 1. A method for increasing the yield of recombinant protein in a microbial fermentation process, which comprises oscillatingly decreasing or increasing the concentration of carbon / energy source in the culture in a short cycle. 2. Method according to claim 1, characterized in that the amplitude is produced by varying the feed rate of the feed solution containing the carbon / energy source. 3. A method according to claims 1 and 2, characterized in that the maximum duration of one cycle is 4 minutes and the duration of a single phase of this cycle is a maximum of 2 minutes. 4. The method according to claims 1 to 3, characterized in that the duration of one cycle is 1 minute and the duration of the phases of this cycle is at most 75% of the total cycle time.
The method described in any one of. 5. A carbon / energy source is added to the culture so as to periodically change the addition rate of the substrate solution for a certain part of the process.
4. The method according to any one of 3 to 3. 6. Process according to claim 1, characterized in that the feed rate is controlled by the cyclic activation and deactivation of the addition of the feed solution. 7. The method according to claim 1, wherein glucose, glycerol, lactose, galactose, methanol, acetate, molasses or starch is used as carbon / energy substrate. 8. Depending on the promoter used, IPTG or indolyl acrylic acid (IAA), or lactose, arabinose, galactose, or methanol to induce the production of recombinant products (already used as an energy source. The method according to any one of claims 1 to 7, characterized in that (if not present) is added to the culture. 9. The method according to any one of claims 1 to 9, characterized in that a temperature change occurs upon induction of the production of recombinant products.